Methods of constructing biodiverse gene fragment libraries and biological modulators isolated therefrom

Abstract

The present invention provides novel methods for producing nucleic acid fragment libraries that express highly diverse peptides or protein domains and, in particular, methods for producing nucleic acid fragment libraries wherein the nucleic acid.

Description

RELATED APPLICATION DATA[0001]This application is a 371 of PCT / AU2004 / 000214 filed Feb. 20, 2004 which claims priority to Ser. No. 10 / 372,003 filed Feb. 21, 2003 and which is a continuation-in-part application of U.S. Ser. No. 09 / 568,229 filed May 5, 2000 which claims the benefit of priority under 35 USC §119(e) from U.S. Provisional Application No. 60 / 132,711 filed May 5, 1999, each of which are herein incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates generally to methods for the production and of nucleic acid fragment libraries that express highly diverse peptides, polypeptides or protein domains and, in particular, methods for producing nucleic acid fragment libraries wherein the nucleic acid fragments of the libraries are derived from one and preferably from two or more prokaryote genomes or compact eukaryote genomes, such as, for example, organisms having diverse characterized genomes. In another embodiment, the nucleic acid f...

AI Technical Summary

Benefits of technology

"The present invention is based on the understanding that peptides in nature have non-random distributions of amino acids and that random peptide libraries have a low frequency of native peptide conformational structures or secondary structures. The inventors have taken advantage of this knowledge by using well-characterized prokaryotic and eukaryotic genomes to control the diversity of peptides expressed in their expression libraries. These libraries have a high degree of diversity in peptides that bind to a target protein or nucleic acid. The invention also involves screening the libraries for bioactive peptides or proteins that have the same activity as they would in their native environment. The invention is particularly useful for identifying novel bioactive peptides or proteins that can be used for drug development."

Problems solved by technology

Moreover, the expressed peptides often show little or none of the secondary or tertiary structure required for efficient binding activity, and / or are unstable.

The relatively unstructured ‘linear’ nature of these peptide aptamers also leads to their more rapid degradation and clearance following administration to a subject in vivo, thereby reducing their appeal as therapeutic agents.

However, the expression libraries described in U.S. Pat. No. 6,319,690 show limited diversity, because the amplified fragments were all antibody-encoding fragments derived from a single complex eukaryote.

However, total normalization of each organism within such uncharacterized samples is difficult to achieve, thereby reducing the biodiversity of the library.

In cases where the environmental sample includes a dominant organism, there is likely to be a significant species bias that adversely impacts on the sequence diversity of the library.

Furthermore, as many of the organisms found in such samples are uncharacterized, very little information is known regarding the constitution of the genomes that comprise such libraries.

Accordingly, it is not possible to estimate the true diversity of such libraries.

Additionally, since the Diversa Corp. process relies upon PCR using random primers to amplify uncharacterized nucleic acids, there is no possibility of accounting for biasing factors, such as, for example, a disproportionate representation of repeated sequences across genomes of the organisms in the environmental sample.

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